Acylated steroids and method of preparing the same



United States Patent 3,002,010 U ACYLATED STEROIDS AND METHOD OF PREPARING THE SAME Victor Emil (Brigoni, Emerson, N.J., and Sidney Fox,

Spring Valley, and Leland Leroy Smith, New City, N.Y., assignors to American Cyanamid Company, New York, N.Y., a corporation of Maine No Drawing. Filed June 18, 1958, Ser. No. 742,743 6 Claims. (Cl. 260397.45)

This invention relates to new steroid compounds. More particularly, it relates to acylated pregnenes and pregnadienes and methods of preparing the same.

It is known that the llp-hydroxyl group of steroids could not be acylated at room temperature using acetic anhydride and pyridine under ordinary conditions and that more drastic treatment such as refluxing (with acetic anhydride in pyridine) usually tended to destroythe hydroxyl group [lourn Am. Chem. Soc. 75, pages 5486- 5489 (1953)]. Successful 'acylations at the llB-position of the steroid nucleus have been carried out in the presence of condensing agents such as toluene-sulfonic acid.

We have now found that the llfi-hydroxy-group (of corticosteroid hormones) can be acylated under controlled conditions as described hereinafter. The compounds prepared by the process of the present invention can be illustrated by the following general formula:

in which R is a lower alkanoyl radical, R and R are hydrogen or lower alkanoyl radicals, R is hydrogen or a lower alkanoyloxy radical, X is hydrogen or a halogen atom and C --C is a divalent radical such as CH=CH-- or CH CH radical.

We have also found that the l7ix-hydroxy group may be acylated using the same conditions as those used for the llfi-hydroxyl provided that the steroid nucleus is also substituted in the l6-position. An example of this is the fact that the 17a-hydroxy group in hydrocortisone will not acylate under the conditions described. However, with l6u-hydroxy hydrocortisone, the acylating will take place using the process of the present invention. Under the usual conditions of acylation, the l7u-hydroxy group does not react with or without the presence of the 160:-

ydroxygroup.

The compounds ofthe present invention are relatively high melting solids. They are somewhat soluble in most organic solvents and relatively insoluble in Water.

The starting material for the preparation of the compounds of the present invention are compounds such as those described in United States Patent No. 2,789,118. These and similar compounds can also be prepared by other methods of inserting the A bond, such as the use of a species of the genus Nocardia, such as Nocardia carollina and also the use of chemical means such as selenium dioxide. The starting compounds of the present invention such as those described in United States Patent No. 2,789,118 are treated with an acylating agent such as a lower aliphatic acyl auhydride under usual conditions, when stoichiometric quantities of acylating agent are used at room temperature, diacetates such as 16,21- diacetates are formed, and the l7m-hy'droxyl group and the Patented Sept. 26, 1951 llfi-hydroxyl group are not infected. In fact, more than stoichioinetric quantities of acylating agent at room temperature will not acylate the ll'lfl-hydroxyl group. How ever, as in the present process, when the acylating reaction is forced by the use of large volumes of acylating agent such as 10 to 30 times the stoichiometric amount, longer periods of contact such as 16-32 hours, elevated temperatures such as 60100' C., mixtures of di, tri, and tetra-acetates are obtained. The separation of these mixtures as shown hereinafter in the examples results in pure 11/3,l6a,21-triacetates and the like. These latter steroids on hydrolysis will produce the corresponding llfl-monoacetates.

Usually the corticosteroid hormones exhibit both glucocorticoid and mineralocorticoid activities. It is, therefore, surprising and unexpected that acylating of the 1113- hydroxyl group in the steroids of the present invention produces compounds having mineralocorticoid activity without any substantial glucocorticoid activity. The mineralocorticoid activity is desirable physiologically since it produces the effect of diuresis and sodium excretion useful in the treatment of edema and similar conditions.

The 11 8- and the l7fi-acylated steroids of the present invention are also useful as intermediates in the synthesis of various compounds having important physiological activity. Transformations with the groups present can be carried out which are otherwise not possible and the protective groups removed subsequently to produce desirable steroids.

In the present application, lower aliphatic acylating agents are those having one to four carbon atoms in addition to the carbonyl group. The definition of the term halogen is intended to include bromine, iodine, chlorine and fluorine. The term flower alkanoyloxy is intended to include those derivatives of alkanoic acid having one a to four carbon atoms in addition to the carbonyl group.

. The following examples describe the preparation of the compounds of the present invention in greater particularity and are intended to be by way of illustration and not limitation.

l1,6,l7a-dihydroxy-1,4-pregnadiene-3,20-dione is dissolved in milliliters of pyridine, and 20 millliters of acetic anhydride is added. The solution is heated at 80 C.- C. for twenty hours. At the termination of the reaction period, the unreacted acetic anhydride is quenched with forty milliliters of methyl alcohol, and the solution is evaporated to an oily residue. This residue is dissolved in milliliters of ethyl acetate and filtered through activated magnesium silicate to remove color. The filtrate is washed with two 25-milliliter portions of a saturated solution of sodium bicarbonate, followed by two 25-milliliter portions of saturated saline solution. The ethyl acetate extract is then dried over anhydrous magnesium sulfate, and the solution is evaporated to dryness.

The crude product was chromatographed through a GO-gram diatomaceous earth column using a solvent system containing twelve parts of toluene, eight parts of petroleum ether (30--60 C.), l3'parts of methyl alcohol and seven parts of water. Twenty-six 25-milliliter cuts Cuts 2-6 are recrystallized from methyl alcohol, yield." I

3 ing 724 mg. of 1118,16a,1704,21-tetraacetoxy-9u-fluoro-1,4- pregnadiene-3,20-dione, having the following properties: melting point 217-219 C.; infrared spectraztetraacetate indicated;

A LOO-gram portion of 160:,21-diacetoxy-9a-fluorol113,l7a-dihydroxy-1,4-pregnadiene-3,20-dione is dissolved in 40 ml. of pyridine and 10.0 ml. of acetic anhydride is added. The solution is heated at 80-85 C. for twenty hours. At the end of the reaction period, a paper chrmatogram shows three components: R (Syst. I) =0.40, 0.52 and 0.71. The unreacted acetic anhydride is quenched with twenty milliliters of methyl alcohol, and the solution is evaporated to an oily residue. This residue is dissolved in 100 milliliters of ethyl acetate and filtered through activated magnesium silicate to remove color. The filtrate is washed with two 25-milliliter portions of a saturated solution of sodium bicarbonate, followed by two 25-milliliter portions of saturated saline solution. The ethyl acetate extract is then dried over anhydrous magnesium sulfate, and the solution is evaporated to dryness. The crude product is chromatographed through a 60-gram diatomaceous earth column using a solvent system containing twelve parts of toluene, eight parts of petroleum ether (3060 C.), 13 parts of methyl alcohol and seven parts of water. Twenty-five 25-milliliter cuts are taken:

Cuts RIG) 1-9 0.71 480 mg. Dried. 11-16 0. 62 116 mg. crystallized out on standing. 17-25 0.40

A 100 mg. portion of l15,160:,21-triacetoxy-9a-fluorol7a-hydroxy-1,4-pregnadiene-3,20-dione from cuts 1l-16 is recrystallized from aeetonezpetroleurn ether, and yields the following characteristics: melting point 22l223 C.; R (Syst. I) =0.52; infrared spectra: triacetate;

[a] =+99.4 (methyl alcohol).

Analysis.-Calcd for C H O F: (1:62.30; H=6.39; F=3.65; acetyl=24.81. Found: C=62.10; H=6.63; F=3.95; acetyl=25.58.

Example 3 formed are filtered, washed with water, and dried under,

reduced pressure at 40 C. to produce 95.0 mg. of 115- acetoxy-9u-fluoro-16u,17 z-21-trihydroxy 1,4 pregnadiene-3,20-dione. Recrystallization from a methyl alcohol: isopropanol mixture yields 55.9'mg. of pure llfi-acetoxy- 9a-fiuoro-l6a,l7a,21-trihydroxy 1,4 pregnadiene 3,20-

dione with the following properties: melting point 228 230 C:; R,(Syst. I)=0.02; infrared spectra: monoacetate;

x53? 236m (e15,250)

Analysis.-Calcd for C H O F: C=63.27; H=6.70; F=4.35. Found: C=63.l0; H=6.90; F=4.10.

To 33 mg. of 1lfl-acetoxy-9a-fiuoro-16a,17a,21-trihydroxy-l,4-pregnadiene-3,20-dione above is added 16 milliliters of acetone and 0.05 milliliter of concentrated hydrochloric acid. The solution is allowed to stand at 25 :5 C. overnight. At the termination of this period, two milliliters of a saturated solution of sodium bicarbonate and five milliliters of water are added to the reaction mixture. The solution is concentrated to approximately seven milliliters under reduced pressure. The product (19 mg.) has the same mobility by paper chromatography as llfi-acetoxy-9a-fluoro-l6u, 17a-isopropylidenedioxy-21- hydroxy-l,4-pregnadiene-3,20-dione.

The product immediately above is dissolved in 0.3 milliliter of pyridine, then 0.01 milliliter of acetic anhydr'ide is added. The solution is evaporated to a residue. Crystallization from methyl alcoholzwater yielded a prodnot which melted at 227-232 C., and which gives an infrared spectrum identical with that of 11 fl-Zl-diacetoxy- 9a-fiuor o-l6a,17a-isopropylidenedioxy-1,4 pregnadiene- 3,20-dione. A mixture melting point shows no depression on admixture of the two compounds.

Example 4 An 810 mg. portion of 9cz-flUOI'O-1lB,l6u17u,21-tetrahydroxy-1,4-pregnadiene-3,20-dione is dissolved in 400 milliliters of acetone and 1.1 milliliter of concentrated hydrochloric acid is added. The solution is allowed to stand overnight at 25: ;5 C. At the termination of this period, it is ascertained by paper chromatogram that the reaction mixture contains 9a-fluoro-l1B,21-dihydroxy-16a, 17a-isopropylidenedioxy 1,4-pregnadiene 3,20 dione (R,(Syst. I)=0.29). Then 50 milliliters of a saturated solution of sodium bicarbonate and milliliters of water are added to the reaction mixture. The solution is concentrated to approximately milliliters and crystallization occurs; the crystals are filtered, washed with water and dried under reduced pressure at 40 C. to obtain 661 mg. of 9a-fluoro-11fi,21-dihydroxy 16a,17a isopropyliden'edioxy-l,4-pregnadiene-3,20-dione. Recrystallization in acetonmpetroleum ether (30-60 C.) yields 620 mg. of the product which melts at 255 -259 C. and has a paper chromatogram R,(Syst. I)=O.29.

A 600 mg. portion of the compound above is dissolved in ten milliliters of pyridine and 2.5 ml. of acetic anhydride is added. The reaction is allowed to proceed at 25i5 C. for two hours. The mixture is then heated for fifteen hours at 90 C. Methyl alcohol (10 ml.) is added to the reaction mixture, and the solution is evaporated to an oily residue. The residue is dissolved in 25 milliliters of ethyl acetate, washed with two 20-milliliter portions of a saturated solution of sodium bicarbonate and two 20-milliliter portions of a saturated saline solution. The ethyl acetate extract is dried over magnesium sulfate, filtered through activated magnesium silicate and evaporated to dryness. The precipitate weighs 700 mg. and has a paper chromatogram R;(Syst. I)=0.92. Three recrystalliz'ations from methyl alcoholzwater and two from acetonezpetroleum ether yields 420 mg. of 11 8,21- diac'etoxy9ii-fluoro-l6a,17oz isopropylidenedioxy 1,4- pregnadiene 3,20-dione having the following properties: melting point 230-232 C.; paper chromatogram R';(Syst. I) =0.92; infrared bands for di-acetate, no hydroxyl; optical rotation [a] =+138 (in methyl alcohol);

mm 236m (e15,300). ana l sisFca'ica for c u o m 0:64.85; H= 6.80; F= 3.66; actyl= 16, 21. Found: 0:64.43; H=7.33; F= 3.97; acetyl=l5.58.

A 170mg, portion of the product immediately. above is dissolved in. l6v ml. of methyl alcoholand' 6.1 milliliters of water plus 2.0 milliliters of concentrated hydrochloric acid are added; The solution is refluxed for threehours. A paper chromatogram showsau. R' (Syst. I)=0.68-. Then fifteen milliliters of water are added to the reaction mixture. The methyl alcohol is evaporated under reduced pressure. Crystallization occurs after allowingto stand at 25i5 C. overnight, yielding 71 mg. of Il-B-acetoxy- 9ci-fluoro-16a,l7u-isopropylidenedioxy 21 hydroxy 1, 4-pregnadiene-3,20-dione havinga paper chromatogram R (Syst. I)=0.71. Two recrystallizations from methyl alcoholzwater resulted in 25.4 mg. of 11fi-acet0xy-9afluoro-l6a,l7'a-isopropylidenedioxy 21 hydroxy 1,4- pregnadiene-3,20-dione having the following properties: melting point 209210' C.; paper chromatogram R (Syst. I)=0.68; infrared bands for monohydroxy, monoacetate;

[a] +128 (0.696% in MeOH).

Analysis.Calcd for C28H33O7FI 0:65.53; H==6.98; F=3.98; acetyl=8.82. Found: .C=65.41; H=7.01; F=4.01; acetyl=l3.13.

Example A 1.00 gram portion of 21-acetoxy-9a-fluoro-11,8,17adihydroxy-4-pregnene-3,20-dione is dissolved in 40 milli-. liters of pyridine and 10.0 milliliters of acetic anhydride added. The solution is heated at 80 85 C. for twenty hours. At the end of the reaction period, a paper ch0 matrograrn shows the following two components: 21- acetoxy-9a fluoro 116,17 dihydroxy-4-pregnene-3,20- dione (Rf 0.48) and 116,21-diacetoxy-9a-fluoro 17ahydroxy 4 pregnene-3,20-dione (Rf Syst. 1:0.70). To the reaction solution, there is added 20 milliliters of methyl alcohol, and the solvents are then removed; leaving an oily residue. This oily residue is dissolved in methyl alcohol, and the solution is filtered through activated magnesium silicate to remove color. The filtrate is evaporated to dryness.

The crude product is chromatographed through a 60- gram diatomaceous earth column using a solvent system, similar to that in Example 1. Seventeen 2-5-milliliter cuts are taken:

Outs R;(Syst.1) I Product 34 0.71 224mg. 8-17 0. 50-0. 72 421 mg. (plus starting material);

ELOH Mai.

236m (el5,800)

Example 6 A 40 ml. portion of methyl alcohol is added to a mixture which contains 11B,16a,2l-triacetoxy-9a-iluoro-17ahydroxy-1,4-pregnadiene-3,20-dione and 11B,16zx,17a,21

tetraacetoxy 90c fluoro 1,4 pregnadiene 3,20 dione. After standing for twenty-five minutes at room temperature (25i5 C.) to allow quenching of the acetic anhydride, the solution is evaporated to dryness. The residue is re-dissolved in 100 ml. of ethyl acetate, filtered through an activated magnesium silicate catalyst and absorbent column and evaporated to dryness. The weight of the crude product is 14.05 grams. The 14.05 grams of impure material is redissolved in 150 ml. of methyl alcohol. The system is evacuated and purged three times with nitrogen gas. A 42.0 ml. quantity of potassium carbonate solution w./v.) is added dropwise over a period of twenty minutes. The reaction is allowed to. continue for an additional fifteen minutes, then 300 ml. of glacial-acetic acid is added to obtain neutral izati'on. Themixture-i's allowed to sand at 0 C. for two hours. The crystals which formed are filtered, Washed with two 100 ml. portions of water and dried under reduced pressure at 40 C. The product is 8.74 grams of 11,8 aeetoxy' 9a; fluoro- 16a,17u,2l trih-ydroxy 1,4-. pregnadiene-3g20-dione. A four-gram portion of the monoacetate is recrystallized twice from acetone: water; and yielded 310- grams of a pure product identified as 904 fluoro l-1;8,16a,17u,2-1 tetrahydroxy 1,4 pregnadiene-3,20-dione.

A 4.7 gram portion of the ll-monoacetate described; above is acetylated' at room temperature (25i5" C.) using 25' ml. of pyridine and 3.0 ml. (2.5 equivalents) of acetic anhydride for a period of six hours. The process,v utilized to extract thev crude product. is identical with that described above, The crude product obtained wasv recrystallized from ethyl acetate: petroleum ether to. yield 3.10 grams of pure material, 11B,16u,2l-acetoxy- 9oz fiuoro 17oz hydroxy 1,4 pregnadiene 3,20- dione: melting point 2 20.5.-.22 3- C.;

x335 23am [wh -P951 (1% in MeOH) Example 7 A 500 mg. quantity of 11,9,16a,17a,21-tetrahydroxy- 4-pregnene-3,20-dione is dissolved in 5 ml. of pyridine, and 2.5. ml. (20 equivalents) of acetic anhydride is added, The mixture is heated at 8085- C. for twenty-six hours.

' The reaction mixture contained a component at At the termination of the reaction period, 10 ml. of methyl alcohol are added to the reaction mixture to quench excess acetic anhydride. The solution is evaporated to dryness. The residue was re-dissolved in 25 ml. of methyl alcohol, filtered through a diatomaceous earth column, concentrated by drying and crystallized from ethyl acetatezpetroleum ether. Weight of crystals119.6 mg. of 115,16u,17a,21-tetraacetoxy-4-pregnene-3,20-dione; melting point210211 C.; molecular weight-546.58; R .(Syst. I) =0.89;

X 239mg (15,900)

[u] +57.4 (1% in methyl alcohol); infrared showed tetraacetate bands and no hydroxyl' band.

Analysis.-Calcd for C H O C, 63.72; H, 7.01; Found: C, 63.48; H, 7.19.

Example 8 The. equivalent of 30 grams of 16d,21dlaC6t OXy'9.0tfluorq 11B,l7a dihydroxy 4 pregnene 3,20 dione is. fermented wit-hthe fungiNocardia carollina in amedium containing an assimilable carbon, nitrogen and mineral salts. The steroids are then removed from the mash by serial extractions with ethyl acetate. The ethyl acetate extracts are pooled and concentrated under reduced pressure. The steroid concentrate is acetylated with 900 ml. of pyridine and 1000 ml. of acetic anhydride for eighteeen hours at room temperature (25:L-5 C.), at which time 1500 ml. of methanol and 1000 ml. of toluene are added and the mixture heated under reduced pressure for four and one-half hours to evaporate off liquids. The acetylated residue contains triamcinolone diacetate l6a,2 l-diacetoxy-9a-fiuoro-1 1B, 17OL-dll1YdI'OXY- l,4-pregnadiene-3,ZO-dione, triamcinolone triacetate (11,8, 1604,21 triacetoxy fluoro 17a hydroxy 1,4- preg-nadiene-3,20-dione) and 16a,2l-diacetoxy-9a-fluoro- 11B,17aadihydroxy-4-pregnene-3,20-dione.

The steroid mixture is partitioned on diatomaceous earth using ,a solvent system composed of ethylene glycol:petroleum etherzmethylene chloride in the proportions 1:4:5. The fraction obtained in the first hold-back volume (HBV) consists of triamcinolone triacetate and lipid impurities. This mixture is concentrated to a mo-.

7 bile oil, washed with water, extracted into ethyl acetate, partially concentrated and petroleum ether is added.

The pricipitated steroid triacetate is filtered, washed with petroleum ether and dried. The steroid material, still crude, weighs 8.55 grams. Partition on diatomaceous earth, using a solvent system composed of ethylene glycokpetroleum etherzmethylene chloride in the proportions 1:10:5, respectively, yields purified triamcinolone triacetate which is recrystallized from methanohpetroleum ether and then acetone: petroleum ether. The product (11fi,16oc,21 triacetoxy 9a fluoro 17m hydroxy- 1,4-pregnadiene-3,20-dione) melts at 190.0191.5 C., resolidifying at 195 C., and remelting (with pre-softening at 218.5 C.) at 221.0223.0 C. (Kofier Block); [a] =+55.4 (in chloroform); R (Syst. I)=0.57;

Example 9 A 150 milligram quantity of11fi,16e,21-triacetoxy-9afiuoro 17a-hydroxy-1,4-pregnadiene-3,20-dione of Example 8 is dissolved in methanol. Nitrogen is bubbled through the solution to displace air. A 14 milligram quantity of sodium metal is dissolved in methanol and deaerated in a similar manner with nitrogen gas. The two solutions are mixed and allowed to stand at room temperature (25-J:5 C.) for ten minutes with a slow stream of nitrogen bubbling through. The reaction mixture is neutralized with acetic acid, extracted with water, and the insoluble residue recrystallized from ethanol. The product is 1lfi-acetoxy-9a-fluoro-16a,17a-21-trihydroxy-1,4-pregnadiene-3,20-dione.

What is claimed is:

1. A process of preparing compounds having the general formula:

(fHgO R3 in which R is a lower alkanoyloxy radical, R and R are members of the group consisting of hydrogen and lower allranoyl radicals, R is a member of the group consisting of hydroxyl and lower alkanoyloxy radicals,

X is a member of the group consisting of hydrogen and a halogen atom and -C --C is a divalent radical of the group consisting of CH=CH-- and -CH --CH radicals which comprises reacting the corresponding 11fihydroxy steroid with an excess of a lower aliphatic acylating agent in the presence of pyridine for from sixteen to thirty-two hours at a temperature within the range of C. to C.

2. A process of preparing 11B,l6a,21-tetraacetoxy- 9a-fluoro-1,4-pregnadiene-3,20-dione which comprises heating 1lB,16a,21-tetrahydroxy 9oz fluoro 1,4-pregnadiene-3,20-dione with an excess of acetic anhydride in the presence of pyridine at a temperature within the range of 60 C. to 100 C. for from sixteen to thirtytwo hours.

3. A process of preparing 11p,16u,21-triacet0xy-9afluoro-17a-hydroxy-1,4-pregnadiene 3,20 dione which comprises heating 16a,21-diacetoxy-11fi,17a-dihydroxy- 9a-fluor0-1,4-pregnadiene-3,20-dione with an excess of acetic anhydride in the presence of pyridine at a temperature within the range of 60 C. to 100 C. for from sixteen to thirty-two hours.

4. A process in accordance with claim 3 in which the triacetate is subsequently hydrolyzed with a member of the group consisting of an alkali metal alkoxide, alkali metal carbonate and a mineral acid to 11B-acetoxy-9afluoro-16a,17a,21 trihydroxy 1,4 pregnadiene 3,20- dione.

5. The compound 11p-acetoxy-9a-fluoro-16u,17a,21- trihydroxy-1,4-pregnadiene-3,20-dione.

6. A compound having the formula:

in which R is a lower allranoyl radical.

References Cited in the file of this patent UNITED STATES PATENTS 2,773,080 Bernstein et al. Dec. 4, 1956 2,774,776 Hogg et al. Dec. 18, 1956 2,781,369 Oliveto et al. Feb. 12, 1957 2,789,118 Bernstein et al. Apr. 16, 1957 2,837,464 Nobile June 3, 1958 2,852,511 Fried Sept. 16, 1958 UNITED STATES PATENT OFFICE CERTIFICATION OF CORRECTION Patent No 3,oo2 o1o September 26,, I961 Victor Emil Origoni ct ale It is hereby certified that error appears in the above numbered patent requiring correction and 'that the saidLetters Patent should read'as corrected below.

Column 6 line 20 for "-acetox read -triacetox column 8, llnes 9 and 11, after "16", each occurrence 1; insert 17a Signed and sealed this 10th day of April 1962.,

(SEAL) Attest:

ERNEST W. SWIDER DAVID L. LADD Attesting Offic r Commissioner of Patents 

1. A PROCESS OF PREPARING COMPOUNDS HAVING THE GENERAL FORMULA: 